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provided by Elsevier - Publisher Connector Cell, Vol. 120, 163–166, January 28, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.01.002 The Spanish Connection: Meeting Review and mRNA Processing Get Even Closer

Manuel Ares, Jr.1,* and Nick J. Proudfoot2,* place before elongation can fully proceed (Figure 1A). 1Department of Molecular Cell At early elongation, the C-terminal domain (CTD) of the and Developmental Biology Pol II large subunit (Rpb1) becomes phosphorylated on University of California, Santa Cruz serine 5 positions within its characteristic heptad re- Santa Cruz, California 95064 peats. Ser5 phosphorylation at CTD recruits the mRNA 2Sir William Dunn School of Pathology capping complex, which adds a cap to the initial nascent University of Oxford transcript. Ser5 phosphorylation is also associated with South Parks Road specific nucleosomal marks at the 5Ј end of the gene, Oxford OX1 3RE such as histone H3 lysine 4 trimethylation (Hampsey United Kingdom and Reinberg, 2003). Several presentations dwelt on new aspects of cou- pling during early transcription elongation. John Lis (Cornell University, New York) described the presence The synthesis of mRNA by RNA polymerase II appears of the elongation factor TFIIS on RNA polymerase II that coupled to numerous RNA-processing events, based is paused at the beginning of Drosophila heat shock on physical or functional connections revealed by bio- genes prior to heat induction. During normal elongation, chemical or genetic tests. New findings were pre- TFIIS is thought to promote cleavage of those tran- sented at a recent meeting in Spain that begin to illumi- scripts whose 3Ј ends are misaligned in the polymerase nate the mechanisms underlying the connections active site, thus allowing polymerase to escape tran- between mRNA processing and specific steps in tran- script arrest (Fish and Kane, 2002). A similar scenario scription (initiation, elongation, and termination) as now appears to operate at the heat shock genes where well as recombination. TFIIS-stimulated transcript cleavage is required to allow the stalled Pol II to resume elongation following heat The production of functional messenger RNA in eukary- shock activation (Adelman et al., 2005). Danny Reinberg otes requires not only transcript synthesis by RNA poly- (University of Medicine and Dentistry of New Jersey, merase II (Pol II) but also multiple RNA-processing steps New Jersey) described biochemical analysis of a large such as capping, splicing, polyadenylation, and RNA complex containing both elongation and re- editing. Recent research has shown that many of these modeling factors. Components of this complex specifi- processing steps occur cotranscriptionally, revealing a cally associate with Lys4 trimethylated histone H3, a close association (coupling) between transcription and well-known histone modification found at the transcrip- mRNA processing (Proudfoot et al., 2002). At a recent tion start site (Ng et al., 2003). Interestingly, this complex meeting in Spain (November 7–10, 2004), new findings significantly stimulates in vitro splicing reactions, as were presented that begin to clarify the connections shown by a collaborative experiment with the laboratory between transcription (initiation, elongation, and termi- of Jim Manley (Columbia University, New York). These nation) and messenger RNA processing and even re- results raise the possibility that recruitment of this com- combination. The meeting, titled “Coupling between plex during early elongation may facilitate subsequent Transcription and RNA Processing” and organized by splicing reactions that occur cotranscriptionally. Miguel Beato and Juan Valcarcel, was hosted by the Universidad Internacional de Andalucia at the Sede An- Packaging mRNA into mRNPs during Elongation: tonio Machado, a restored 17th century palace in Baeza, The R Loop Story site of a Renaissance university in the south of Spain. It has long been known that pre-mRNA is packaged into The intimate setting and small size of the meeting suc- protein-associated complexes (mRNPs, originally called cessfully encouraged open discussion. A shared goal of hnRNPs) that contain both general nuclear proteins such the participants was to learn more about the molecular as hnRNP A (and other family members) and more spe- mechanisms coupling transcription and RNA pro- cific splicing/mRNA export-associated protein factors cessing. Such connections are already in part defined by such as the THO/TREX complex (Jensen et al., 2003). protein-protein or protein-nucleic acid interaction data While this packaging process is well known to be re- using either biochemical or genetic tests. However, the quired for mRNA maturation, it also appears to be re- precise mechanisms by which coupling occurs remain quired for protection of the integrity of the DNA template largely mysterious. itself. New findings were presented on the intriguing phenomenon of transcription-dependent hyperrecom- bination, which is stimulated by defects in elongation Early Elongation Coupling factors or in mRNA export. Recently published studies Once Pol II has escaped from a gene promoter into early in yeast from Andres Aguilera’s lab (University of Seville, elongation, a number of molecular events appear to take Spain; Huertas and Aguilera, 2003) suggested a provoc- ative model in which, if the nascent RNA emerging from Pol II is not immediately and properly coated by mRNP *Correspondence: [email protected] (M.A.); nicholas.proudfoot@ packaging proteins, then it will result in the formation pathology.oxford.ac.uk (N.J.P.) of an R loop with underwound DNA located just behind Cell 164

Figure 1. Cartoons Depicting Four Stages in the Coupling of Pol II Transcription with Pre-mRNA Processing See text for full details. (A) Elongating Pol II may be poised at the start of the gene with its CTD heptad repeats in a serine 5-phosphorylated state and the nucleosomes marked by histone H3 lysine 4 trimethylation (H3-K4 trimethylation). The involvement of TFIIS and the capping complex is indicated. (B) Deficient pre-mRNA packaging may result in topological pausing of elongating Pol II through the formation of pre-mRNA:DNA R loops. The looped-out sense DNA strand is then susceptible to DNA damage by hyperrecombination. R loops may be relieved by RNase H activity. (C) Pre-mRNA is cotranscriptionally packaged, spliced, and edited during elongation. Nucleosomes are also repositioned on the DNA chromatin template during elongation. (D) Termination of Pol II transcription. Cleavage and polyadenylation at the 3Ј end of the pre-mRNA is mediated by cleavage/polyadenylation factors. The downstream transcript that remains attached to the elongating Pol II is then subjected to 5Ј-3Ј exonucleolytic degradation, eventually leading to the release of Pol II from the DNA template. the elongating polymerase (Figure 1B). This in turn leads elongation. Jensen showed that mutations in elongation to both impairment of transcription and a hyperrecombi- factors such as Rad3 (a subunit of TFIIH) and Dst1 (a nation phenotype restricted to the transcribed locus. homolog of mammalian TFIIS) mitigated the transcrip- Now Jim Manley’s group reports the unexpected finding tion elongation defect caused by mutations in the THO that genetic inactivation of the SR protein splicing factor complex, suggesting that the inadequacy of mRNP pack- ASF/SF2 in chicken DT40 cells (Wang et al., 1998) results aging caused by lack of THO is more pronounced when in a similar phenotype. This new study highlights the transcription elongation proceeds at a higher rate (Jen- critical importance of properly integrating transcription sen et al., 2004). On a related theme, he also showed that with the correct processing of the nascent transcript. THO defects appear to adversely affect later elongation Together, these findings bolster the hypothesis that de- stages, causing apparent premature termination. Similar fective packaging of the nascent transcript leads to R observations were also made in the Aguilera lab. Manny loop formation behind the polymerase, which stimulates Ares (University of Santa Cruz, California) reported on recombination and may also drag the elongating poly- experiments with Grant Hartzog’s lab in which they merase to a halt. found that yeast cells carrying mutations in genes for The contribution of nascent RNA packaging to elonga- THO/TREX/, PAF, and FACT complex subunits share tion was further illuminated by exciting data from Andres highly similar genome-wide reduction in pre-mRNA and Aguilera and Torben Heick Jensen (Aarhus University, mRNA levels. Both PAF and FACT are chromatin-associ- Denmark). They both described experiments illustrating ated elongation factors (Hampsey and Reinberg, 2003; the effect of THO complex mutations on transcription Belotserkovskaya et al., 2003). This is consistent with Review 165

the notion that compromising the function of proteins Attempts to capture the synchronicity of transcription involved in mRNA packaging and elongation leads to and splicing events in vivo using chromatin crosslinking common downstream defects in gene expression (Bur- of splicing factors continue to move forward in yeast, ckin et al., 2005). All of these results suggest that, in following on the initial finding that the U1 snRNP is the absence of efficient mRNP packaging, topological cotranscriptionally recruited to intron-containing genes problems arise when the nascent RNA exits the elongat- only (Kotovic et al., 2003). That work showed that the ing Pol II complex. First, R loops may form (as described U1 snRNP associates with chromatin via splice donor above), and second, transcription elongation may be site sequences, rather than at promoters or downstream topologically restricted, a problem that could be exacer- regions in general, indicating that its recruitment is spec- bated at the end of highly transcribed genes. ified by introns. New work from Karla Neugebauer’s lab (University of Dresden, Germany) presented by Janina Coupling Splicing to Transcription Elongation Gornemann extends this study by showing that addi- Although mRNA splicing can be shown to occur in test tional components of the splicing machinery also accu- tubes on synthetic RNA substrates, it is likely that splic- mulate on intron-containing genes in a temporal and ing in vivo is tightly regulated (Figure 1C). Indeed, it has spatial pattern consistent with nascent RNA-dependent been demonstrated over recent years that transcrip- recruitment or stabilization. Studies using yeast strains tional elongation can directly influence splicing patterns harboring deletions of conserved but nonessential splic- across larger genes (de la Mata et al., 2003; Proudfoot, ing factors are beginning to provide intriguing informa- 2003). Alberto Kornblihtt’s group (University of Buenos tion on how different factors influence the timing of Aires, Argentina) presented evidence of a polar effect snRNP association with the transcribed gene. for . It appears that the inclusion of an upstream alternative exon enhances the subsequent inclusion of a downstream alternative exon in the same Coupling of Polyadenylation and Editing nascent transcript. This exciting development could to Transcription open the way to understand how remote alternative Connections between transcription and mRNA pro- splicing events might be coordinated within the same cessing are also important at the end of genes. Indeed, transcript, possibly through interactions that occur dur- mRNA 3Ј end processing and polyadenylation have ing elongation. In particular, Kornblihtt described experi- been known to be associated with Pol II termination for ments on the fibronectin gene with either of two alterna- almost two decades. Now, independent studies in both tive exons mutated in heteroallelic transgenic mice yeast and mammalian systems provide new insights into (made in Tito Baralle’s laboratory at ICGEB, Trieste, Italy; this coupling process. Depletion of a nuclear 5Ј-3Ј exo- Chauhan et al., 2004). He showed that the polar effect nuclease (Rat1p in yeast or Xrn2 in human cells) causes occurs in cis and that, although inclusion of the up- a clear termination defect, and data presented by Steve stream exon enhances inclusion of the downstream Buratowski (Harvard Medical School, Boston) and Nick exon, the inclusion of the downstream exon does not Proudfoot (University of Oxford, UK) showed that the influence inclusion of the upstream exon. Both the polar exonuclease acts like a molecular torpedo by attaching effect and its allelic specificity strongly suggest that to the 5Ј end of the nascent RNA generated by cleavage alternative splicing decisions in the upstream parts of either at the poly(A) signal or at cotranscriptional cleav- a transcript can influence those downstream, possibly age sites. In a model that is reminiscent of rho-depen- during transcription elongation. dent termination in bacteria, the 5Ј-3Ј exonuclease tor- More is sure to be learned about the molecular events pedo is thought to track its way along the RNA chain connecting splicing to transcription now that a coupled to the elongating polymerase and trigger destabilization in vitro system is under development. Barbara Natalizio of the ternary complex (Kim et al., 2004; West et al., of Mariano Garcia-Blanco’s laboratory (Duke University, 2004; Teixeira et al., 2004) (Figure 1D). North Carolina) presented her work employing human A final example of coupling is the intriguing process cell extracts that could support both transcription and of mRNA editing (Keegan et al., 2001), which is mani- splicing. Comparison of splicing of exogenously added fested by targeted deamination in mRNAs (cytosine to transcripts generated by T7 polymerase with those syn- uracil and adenosine to inosine). In the later case, adeno- thesized in situ by either added T7 polymerase or the sine deaminases (ADARs) recognize specific RNA du- endogenous RNA polymerase II indicates that in situ- plexes in pre-mRNA, resulting in conversion of selected synthesized Pol II transcripts are more rapidly spliced. adenosine to inosine. Inosine is recognized in the trans- Splicing of a two intron ␤-globin precursor is much more lation process as guanosine, hence changing the ge- efficient in this system than observed previously (Ghosh netic code. Two presentations from Liam Keegan (MRC and Garcia-Blanco, 2000). Several critical modifications Human Genetics Unit, Edinburgh, United Kingdom) and in the composition of the system will be described in a Marie Ohman (Stockholm University, Sweden) reminded forthcoming manuscript. Although it is still not clear how us that mRNA editing sites recognized by the enzyme precisely “cotranscriptional” these splicing events are ADAR are often partly intronic; therefore, in such cases, (the precursor accumulates rapidly, but splicing occurs editing must occur prior to splicing. Furthermore, Marie quite slowly), the system described by Natalizio may Ohman described the requirement of the Pol II CTD for present an attractive alternative to simply tossing in efficient editing at these intron-exon junction sites. Thus, naked T7-derived transcripts for those of us who want akin to capping, splicing, and polyadenylation, the to know how the machinery of transcription anticipates mRNA editing process also appears to be tightly linked the need to splice. to Pol II through the versatile CTD. Cell 166

What Does Coupling Really Mean? DNA:RNA hybrids mediate transcription elongation impairment and Toward the end of the meeting, a panel discussion was transcription-associated recombination. Mol. Cell 12, 711–721. held to address the question, “What does coupling really Jensen, T.H., Dower, K., Libri, D., and Rosbash, M. (2003). Early mean?” While many are happy with the flexibility af- formation of mRNP: license for export or quality control? Mol. Cell 11, 1129–1138. forded by the ambiguity of the word “coupling,” others Jensen, T.H., Boulay, J., Olesen, J.R., Colin, J., Weyler, M., and Libri, felt that specific biochemical or molecular criteria ought D. (2004). Modulation of transcription affects mRNP quality. Mol. to be applied. These criteria may vary and could be Cell 16, 235–244. articulated by applying various adjectives, such as “ki- Keegan, L.P., Gallo, A., and O’Connell, M.A. (2001). The many roles netic coupling,” “tight coupling,” “physical coupling,” or of an RNA editor. Nat. Rev. Genet. 2, 869–878. “informational coupling.” However, no consensus could Kim, M., Krogan, N.J., Vasiljeva, L., Rando, O.J., Nedea, E., be reached before dinner. Everyone did agree that they Greenblatt, J.F., and Buratowski, S. (2004). The yeast Rat1 exo- heard new interpretations of what coupling might be nuclease promotes transcription termination by RNA polymerase II. and how it might be implemented during Pol II transcrip- Nature 432, 517–522. tion. The notion that the CTD is central to the coupling Kotovic, K.M., Lockshon, D., Boric, L., and Neugebauer, K.M. (2003). process, while still not proven, was not significantly di- Cotranscriptional recruitment of the U1 snRNP to intron-containing genes in yeast. Mol. Cell. Biol. 23, 5768–5779. minished, either. Regardless of the precise role of the CTD, the focus is clearly on the events that occur on Ng, H.H., Robert, F., Young, R.A., and Struhl, K. (2003). Targeted recruitment of Set1 histone methylase by elongating Pol II provides the nascent transcript as it emerges from the polymer- a localized mark and memory of recent transcriptional activity. Mol. ase and on how these molecular events are coordinately Cell 11, 709–719. managed. The events that trail behind the elongating Proudfoot, N.J. (2003). Dawdling polymerases allow introns time to polymerase, including capping, mRNP assembly, chro- splice. Nat. Struct. Biol. 10, 876–878. matin reassembly, efficient splicing, alternative splicing, Proudfoot, N.J., Furger, A., and Dye, M. (2002). Integrating mRNA polyadenylation, editing, and termination, exert diverse processing with transcription. Cell 108, 501–512. impacts both on the transcript structure and on polymer- Teixeira, A., Tahiri-Alaoui, A., West, S., Thomas, B., Ramadass, A., ase function. It is safe to anticipate that there will be Martianov, I., Dye, M., James, W., Proudfoot, N.J., and Akoulitchev, new surprises in this area in the near future. A. (2004). Autocatalytic RNA cleavage in the human beta-globin pre- mRNA promotes transcription termination. Nature 432, 526–530. Wang, J., Xiao, S.H., and Manley, J.L. (1998). Genetic analysis of the Acknowledgments SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing. Genes Dev. 12, 2222–2233. We are grateful to our colleagues for allowing their unpublished Ј → Ј work to be described. We apologize to participants at the meeting West, S., Gromak, N., and Proudfoot, N.J. (2004). Human 5 3 whose work we were unable to include in this account of the Baeza exonuclease Xrn2 promotes transcription termination at co-tran- meeting. Finally, we are indebted to the meeting organizers and the scriptional cleavage sites. Nature 432, 522–525. Universidad Internacional de Andalucia Sede Antonio Machado for a unique scientific and Spanish experience.

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